We propose to develop non-toxic preclinical approaches for improving alloengraftment in patients sensitized by transfusion therapy using non-myeloablative conditioning regimen and conventional marrow cell dose. We noted that 2 Gy TBI permits uniform engraftment in rodents if anti CD154 mAb is infused. A major challenge continues to be engraftment of sensitized recipients given unrelated rather than matched sibling donor transplants. We have two basic hypotheses to explain why sensitized recipients are more likely to reject donor BM grafts: 1. T cell clonal size of host anti-donor T cells is substantially larger; 2. Costimulatory pathway dependency and requirements for memory host anti-donor alloreactive T cell expansion and function differ from those required for naive T cells. To overcome sensitization, we will need distinct types of approaches. The first will seek to reduce host anti-donor clonal T cell size by administering fludarabine or rapamycin. Because sufficient numbers of T cells may escape these pharmacological approaches, we will test cellular therapies to eliminate (veto) or suppress (CD4+25+) host anti-donor alloreactive T cell expansion. Although these approaches will reduce the graft rejection pool size, both effector/memory and newly generated naive T cells with alloreactive capacities will be able to respond to new antigenic challenge in the form of donor BM cells. Therefore, we will explore strategies to target T cell costimulatory pathways that may affect T cell expansion and/or function. We have developed new models to simultaneously track naive and effector/memory cell responses and alloantigen-specific CD4+ and CD8+ T cells capable of BM rejection. These studies will provide new insights into the immunobiology of graft rejection in sensitized recipients and led to innovative strategies to inhibit graft rejection that ultimately can be translated into the clinic.